The glycosyl-phosphatidylinositol (GPI)-anchored folate receptor (FR) binds folate compounds and folate conjugates and mediates their uptake by cells. FR-mediated transport is believed to occur via a novel sphingolipid-rich membrane microdomain that also contains other GPI-anchored proteins and that recycles between the cell surface and endocytic compartments, but the molecular components of this transport machinery have not been identified. We propose to identify genes whose products are essential for FR recycling and the associated folate uptake by genetic analysis in a yeast system. The system is chosen because of its versatility and based on the close similarity in known membrane transport mechanisms between yeast and mammalian cells as well as the similarity in membrane-associated characteristics of yeast and mammalian GPI-anchored proteins. We will use a yeast folate auxotroph in which folate uptake (presumably by diffusion) requires a very high (about 100 microM) exogenous folate concentration. We have introduced human FR into this yeast strain under control of a Cu++-inducible promoter and found that the receptor mediates [3H]folic acid uptake at nanomolar extracellular concentrations, supports cell growth in less than 0.1 microM folinic acid and sensitizes the cells to low concentrations of the potent antifolate drug, dideazatetrahydrofolate (DDATHF). We will mutagenize the FR expressing yeast chemically or by transposon insertion and initially select for mutants that are resistant to low concentrations of DDATHF. A second more stringent replica plating screen will narrow mutants defective in FR-mediated transport by selecting for those that require a high (about 100 microM) concentration of exogenous folinic acid for normal growth. The transport defects will be confirmed by [3H]folic acid uptake studies. Alternatively, temperature-sensitive mutants defective in folate uptake will also be isolated. FR mutants, as well as mutants with impaired FR synthesis or GPI-modification, will not be considered. A yeast genomic library and a human cDNA expression library will be used to complement the transport defects. The yeast and human genes complementing the mutant phenotypes as well as the yeast mutations will be examined by DNA sequence analysis. For the yeast genes thus identified, putative human homologs will be tested for complementation of mutant phenotypes. The mechanistic roles of such known and novel proteins in FR-mediated transport will be the subject of future investigations. The studies are expected to provide new insights of a fundamental nature into membrane transport processes.